The present invention relates generally to inosine 5xe2x80x2-monophosphate dehydrogenase (IMPDH) and more particularly to purified and isolated DNA sequences that encode proteins possessing the biological properties of eukaryotic IMPDH, to the expression products of these DNA sequences in transformed or transfected host cells, to recombinant and synthetic proteins and peptides having amino acid sequences based on the sequence of amino acids deduced from these DNA sequences, to antibodies specific for such proteins and peptides, to analytical procedures for the detection and quantification of such peptides and proteins and nucleic acids related thereto, to the use of IMPDH-encoding DNA sequences as selectable markers and as tools for gene amplification in recombinant hosts, and to cell lines and organisms displaying enhanced production of IMPDH and/or elevated levels of products such as guanosine monophosphate (GMP), whose synthesis in cells is dependent on the activity of IMPDH.
The enzyme IMPDH (EC 1.2.1.14) catalyzes the formation of xanthine monophosphate (XMP) from inosine monophosphate (IMP). In the purine de novo synthetic pathway, IMPDH is positioned at the branch point in the synthesis of adenine and guanine nucleotides and is thus the rate-limiting enzyme in the de novo synthesis of guanine nucleotides, such as guanosine 5-monophosphate. [Weber, Cancer Res., 43:3466-3492 (1983); Weber, et al., Adv. Enzyme Regul., 18:3-26 (1980)]. Inhibition of cellular IMPDH activity results in an abrupt cessation of DNA synthesis [Franklin, et al., Biochem. J., 113:515-524 (1969); Cohen, et al., J. Biol. Chem., 256:8713-8717 (1981); and Duan, et al., Cancer Res., 47:4047-4051 (1987)] and a cell cycle block at the Gl-S interface [Cohen, et al., Cancer Res., 43:1587-1591 (1983)]. Because IMPDH is essential in providing the necessary precursors for DNA and RNA biosynthesis, normal tissues that exhibit increased cell proliferation generally exhibit increased IMPDH activity [Jackson, et al., Nature, 256:331-333 (1975); Jackson, et al., Biochem. J., 166:1-10 (1977), Cooney, et al., Anal. Biochem., 130:339-345 (1983)]. Similarly, increased cell proliferation is accompanied by elevated enzyme activity in certain rat hepatomas with varied growth rates. Weber, Cancer Res., 43:3466-3492 (1983). These hepatomas manifest IMPDH activities that are disproportionately higher than those of normal tissues, suggesting that IMPDH is associated with cell proliferation and may be linked to either malignant cell transformation or tumor progression.
To investigate the role of IMPDH in growth regulation and malignancy, attempts have been made to purify the enzyme to homogeneity to allow preparation of specific antibodies thereto and to isolate IMPDH-encoding DNA.
IMPDH isolated from bacterial sources has been determined to vary widely with respect to allosteric properties, size, and subunit composition. IMPDH isolated from E. coli has been purified and characterized as a tetramer of identical subunits [Gilbert, et al., Biochem. J., 183:481-494 (1979); and Krishnaiah, Arch. Biochem. Biophys., 170:567-575 (1975)]. Unlike mammalian cell enzymes, the E. coli IMPDH enzyme is reported to be insensitive to the inhibitory effect of mycophenolic acid [Franklin, et al., Biochem. J., 113:515-524 (1969)]. In E. coli, IMPDH has been determined to be the product of the guaB locus and the sequence of the quaB structural gene and surrounding DNA has been determined to span 1.533 kb and to code for an IMPDH subunit sequence of 511 amino acids with a calculated molecular weight 54,512 [Tiedeman, et al., Nucleic Acids Research, 13:1303 (1985)].
Miyagawa, et al., Bio/Tech., 4:225 (1986), have described the cloning of the Bacillus subtilis IMPDH gene, which, upon re-introduction into a B. subtilis strain that overproduced inosine, resulted in an increased production of guanosine, accompanied by a decreased accumulation of inosine. The IMPDH gene was localized on a 6.5 kb insert and further localized to a Hind III-partially digested 2.9 kb fragment. However, the gene was not reported to have been isolated and no information was provided with respect to the DNA sequence of the gene.
While a number of workers have reported the purification or partial purification of IMPDH from a variety of eukaryotic cell sources, including ascites cells, thymus cells, mouse LS cells, and other mammalian cells, none have been successful in obtaining substantial information about the amino acid sequence of the IMPDH protein, or in establishing the utility of anti-IMPDH antibodies in the characterization of the cellular role of IMPDH.
Eukaryotic IMPDH has been obtained from one plant and several animal species, including cowpea nodule cells [Atkins, et al., Arch. Biochem. Biophys., 236:807-814 (1985)], Yoshida sarcoma ascites cells [Okada, et al., J. Biochem., 94:1605-1613 (1983)], rat hepatoma 3924A cells [Ikegami, et al., Life Sci., 40:2277-2282 (1987) and Yamada, et al., Biochem., 27:2193-2196 (1988)] and Chinese hamster cells [Collart, et al., Mol. Cell. Biol., 7:3328-3331 (1987)]. The disclosures of the last-mentioned publication by the present inventors are specifically incorporated by reference herein. In all of these reports, denaturing polyacrylamide gel electrophoresis was used to assess purity and to estimate molecular weight. The reported molecular weight for all of the above mentioned enzymes was approximately 56,000. A polyclonal antibody raised against the purified protein was prepared for the enzyme isolated from Yoshida sarcoma ascites cells, rat hepatoma 3924A cells, and Chinese hamster cells. As described in detail, infra, only in the case of the antibody prepared against the Chinese hamster enzyme was an antibody determined to be useful in examination of cellular regulation and useful in isolation of eukaryotic IMPDH-encoding DNA.
There continues to exist a need in the art for information regarding IMPDH enzymes of eukaryotic origins (especially of vertebrate and more particularly of mammalian origins) such as can be provided by the isolation, sequencing, and recombinant system utilization of DNA sequences encoding the same. The availability of such materials and information would make possible a vast array of novel systems and methodologies based thereon including methods and materials useful in production of products displaying IMPDH activity.
The present invention provides novel purified and isolated DNA sequences encoding eukaryotic inosine 5xe2x80x2-monophosphate dehydrogenase (IMPDH), which have allowed for the initial determination of the primary structural conformation (i.e., amino acid sequence) of the eukaryotic protein. Specifically provided are sequences encoding human, mouse, and Chinese hamster IMPDH. Provided also are alternate DNA forms such as genomic DNA and DNA manufactured by partial or total chemical synthesis from nucleotides. The association of DNA sequences of the invention with expression regulatory DNA sequences, such as promoters, enhancers and the like, allows for in vivo and in vitro transcription to form messenger RNA, which, in turn, is subject to translation to provide IMPDH protein in large quantities.
Among the multiple aspects of the present invention, therefore, is the provision of (a) novel eukaryotic IMPDH DNA sequences as set out in FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 3A, FIG. 3B, and FIG. 3C; (b) IMPDH-encoding DNA sequences, which hybridize thereto under hybridization conditions of the stringency equal to or greater than the conditions described herein as used in the initial isolation of cDNAs of the invention, which also encode proteins with IMPDH biological activities; and (c) DNA sequences encoding the same, allelic variant and/or analog IMPDH proteins, which incorporate, at least in part, degenerate codons. Correspondingly provided are viral or circular plasmid DNA vectors incorporating such DNA sequences and prokaryotic and eukaryotic host cells transformed or transfected with such DNA sequences and vectors, as well as novel methods for the recombinant production of IMPDH proteins through cultured growth of such hosts and isolation thereof from the hosts or from their culture media.
According to another of its aspects, cell lines and organisms having enhanced production of IMPDH, as well as enhanced production of guanosine monophosphate (GMP), are also provided. Preferred embodiments of such cells include the transformed or transfected host cells described initially above. Also comprehended are naturally occurring or mutagenized eukaryotic cells, which are selected for increased IMPDH production (e.g., on the basis of capacity for growth in the presence of elevated levels of cytotoxic IMPDH inhibitors) and then additionally subjected to stepwise incremental selection in the presence of a cytotoxic IMPDH inhibitor such as mycophenolic acid (MPA), ribavirin, brenidin, and tiazofurin. Illustratively, naturally occurring or mutagenized cells capable of growing in medium containing 0.1 to 0.5 xcexcg/mL MPA are subjected to stepwise selection at increasingly higher levels of the agent.
The preparation and incorporation of IMPDH DNA sequences for use as a selectable marker to select for cells that have incorporated a selected fragment of foreign DNA into their genetic material is also embraced by the present invention. In one illustration of the DNA selection systems of the invention, Chinese hamster IMPDH encoding DNA is operatively associated in a plasmid construct with appropriate expression control sequences, and e.g., a DNA sequence coding for the E. coli gpt protein. This plasmid construct is then introduced into hamster cells, and cells functionally incorporating the IMPDH/gpt gene construct are selected on the basis of survival in culture media that contains MPA.
Novel protein products of the invention include recombinant-produced compounds having the primary structural conformation (i.e., amino acid sequence) of IMPDH protein, as well as peptide fragments thereof and synthetic peptides assembled to be duplicative of amino acid sequences thereof. Proteins, protein fragments, and synthetic peptides of the invention are projected to have numerous uses including therapeutic, diagnostic and prognostic uses and also provide the basis for the preparation of monoclonal and polyclonal antibodies specifically immunoreactive with IMPDH. Preferred protein fragments and synthetic peptides include those that duplicate continuous antigenic epitope sequences of the full-length protein.
Preferred protein products of the invention include approximately 56 kDa IMPDH peptides having the deduced sequence of 514 amino acid residues for human and Chinese hamster proteins set out in FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D.
The preferred 56 kDa IMPDH polypeptide is characterized by a capacity to specifically bind IMP with a Ki equal to approximately 25 xcexcmol, a sensitivity to inhibition by IMPDH inhibitors such as mycophenolic acid, and immunoprecipitability by rabbit anti-IMPDH antisera.
Antibodies specific for the novel peptide products of the invention preferably bind with high immunospecificity to IMPDH protein, fragments, and peptides, recognizing eptitopes that are not common to other proteins.
Also provided by the present invention are novel procedures for the detection and/or quantification of the IMPDH protein, as well as the corresponding nucleic acids, e.g., DNA and mRNA, associated therewith. Antibodies of the invention may be used in known immunological procedures for quantitative detection of IMPDH protein in fluid and tissue samples. DNA sequences of the invention may be suitably labeled and used for the quantitative detection of mRNA encoding IMPDH or assessment of any genetic alteration resulting in amplification or rearrangement of the IMPDH gene.